PIM2_LanthaScreen_Binding

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LanthaScreen™ Eu Kinase Binding 
Assay Validation Packet 
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Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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LanthaScreen™ Eu Kinase Binding Assay for PIM2 
Overview 
This protocol describes how to perform a LanthaScreen™ Eu Kinase Bindng Assay designed to detect and characterize 
kinase inhibitors. Procedure 1 describes an experiment to optimize the concentration of tracer to use with a specific kinase 
target. Procedure 2 describes how to perform kinase inhibitor affinity (IC50) measurements, using either the concentration of 
tracer determined by the user following Procedure 1 or using the concentration determined experimentally by Invitrogen. 
The protocol is accompanied by representative data generated at Invitrogen for both procedures. 
 
LanthaScreen™ Eu Kinase Binding Assays are based on the binding and displacement of a proprietary, Alexa Fluor® 647-
labeled, ATP-competitive kinase inhibitor scaffold (kinase tracer) to the kinase of interest (Figure 1). Tracers based on a 
variety of scaffolds have been developed in order to address a wide range of kinase targets. Binding of the tracer to the 
kinase is detected using a europium-labeled anti-tag antibody, which binds to the kinase of interest. Simultaneous binding of 
both the tracer and antibody to the kinase results in a high degree of FRET (fluorescence resonance energy transfer) from the 
europium (Eu) donor fluorophore to the Alexa Fluor® 647 acceptor fluorophore on the kinase tracer. Binding of an inhibitor 
to the kinase competes for binding with the tracer, resulting in a loss of FRET. 
 
Because the kinase tracers are based on ATP-competitive kinase inhibitors, they are suitable for detection of any compounds 
that bind to the ATP site. This includes “Type II” inhibitors (e.g. Gleevec®/imatinib, sorafenib, BIRB-796), which bind to 
both the ATP site and a second site often referred to as the “allosteric” site. 
 
In contrast to most fluorescence-based kinase activity assays, LanthaScreen™ Eu Kinase Binding Assays can be read 
continuously, which facilitates evaluation of compounds with slow binding kinetics. Also, unlike most activity assays, the 
assay can be performed using either active or non-activated kinase preparations, which enables characterization of 
compounds that bind preferentially to non-activated kinases (e.g. Gleevec®/imatinib). 
 
Figure 1. Schematic of LanthaScreenTM Eu Kinase Binding Assay 
 
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Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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Materials required 
 
Product Name Part Number Concentration Quantity Notes
PIM2 PV3649 0.1 to 0.5 mg/mL 10 μg (1) 
5X Kinase Buffer A PV3189 5X 4 mL (2) 
Kinase Tracer 236 PV5592 50 μM in DMSO 25 μL (3) 
LanthaScreen™ Eu-anti-GST Antibody PV5594 or PV5595 0.22 to 0.28 mg/mL (1.5 to 1.8 μM) 25 μg or 1 mg (4) 
Staurosporine (optional) PHZ1271 N/A 100 μg (5) 
 
(1) PIM2 is supplied at a concentration between 0.1 to 0.5 mg/mL, with the exact concentration is printed on the product 
label. The molecular weight of the kinase is 63.7 kD, which can be found on the kinase Certificate of Analysis 
shipped with the product or at Invitrogen.com/kinase. The kinase molecular weight will be needed to convert the 
concentration to molarity as required in the following protocol. 
(2) Kinase Buffer A is supplied as a 5X concentrated stock. Prepare a 1X solution by adding 4 mL of the 5X solution to 
16 mL of distilled H2O. The 1X kinase reaction buffer is stable at room temperature. 1X Kinase Buffer A consists of 
50mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35. 
(3) Kinase Tracer 236 is supplied as a 50 μM stock in DMSO. 
(4) Prior to use, the antibody tube should be centrifuged at approximately 10,000 x g for 5 minutes, and the solution 
needed for the assay should be aspirated from the top of the solution. This centrifugation step will eliminate spurious 
data points that can arise on occasion due to any particulates in the product. 
(5) A 1 mM stock of staurosporine can be prepared by dissolving 100 μg of staurosporine in 210 μL of DMSO. 
 
 
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Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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Basic protocol for inhibitor studies 
LanthaScreenTM Kinase Binding Assays to evaluate inhibitors are typically performed by addition of 3 components each at 
3X the final desired concentration as follows: 
 
1. Add 5 µL of test compound. 
2. Add 5 µL of kinase/antibody mixture. 
3. Add 5 µL of tracer. 
4. Incubate for 1 hour at room temperature and read plate. 
 
Final assay conditions for inhibitor studies 
5 nM kinase1 
2 nM Eu-Anti-GST Antibody 
100 nM Kinase Tracer 2362 
1X Kinase Buffer A 
 
1A kinase concentration of 5 nM kinase is recommended as a starting point for assay development as it typically 
results in a robust signal. Decreasing the kinase concentration may be necessary for accurate measurement of very 
tight-binding inhibitors, similar to kinase activity assays. For specific test cases, successful assays have been 
performed with at little as 200 pM kinase, though the assay window may be lower. 
2A tracer concentration of 100 nM is suggested by Invitrogen, but could also be experimentally determined or 
optimized in Procedure 1. Optimal tracer concentrations for all validated kinases typically fall within the 1 to 100 
nM range. 
 
 
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Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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Plate readers 
The data presented in this document were generated using a Tecan Infinite F-500 plate reader using the appropriate filters and 
instrument settings for europium-based LanthaScreen™ assays. The assay can be performed on a variety of plate readers 
including those from Tecan (Ultra, Infinite F-500, Safire2), Molecular Devices (Analyst and M5), BMG LABTECH 
(PHERAstar) and Perkin Elmer (EnVision, Victor, and ViewLux) or any other plate reader configured for LANCE® or 
HTRF® assays. General instrument settings are listed in the table below: 
 
Excitation 340 nm (30 nm bandpass) 
Kinase Tracer Emission 665 nm (10 nm bandpass) 
LanthaScreen™ Eu-anti-Tag Antibody 
Emission 615 nm (10 nm bandpass) 
Dichroic Mirror Instrument dependent 
Delay Time 100 µs 
Integration Time 200 µs 
 
For additional assistance, ask your Invitrogen representative for instrument-specific setup guidelines, or contact Invitrogen 
Discovery Sciences technical support at 800-955-6288 (select option 3 and enter 40266), or email 
drugdiscoverytech@invitrogen.com for more information on performing these assays on your particular instrument or for a 
control to test an instrument. 
Plates 
Assays are typically performed in black, low-volume 384-well plates (Corning Part#3676). However, white plates may yield 
higher quality data (Corning Part#3673) for assays that give a relatively low assay window (1.5 to 2 fold) that are being 
measured on monochromater-based instruments or older filter-based instruments. For most kinases and most plate readers, 
plate color has littleor no impact. 
 
 
 
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Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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Procedure 1. Optimization of tracer concentration 
This step describes how to optimize the tracer concentration for use in subsequent inhibitor studies by performing binding 
assays with a 2-fold serial dilution of tracer. This experiment allows for approximation of the tracer dissociation constant 
(Kd) and evaluation of the signal strength or “assay window” as a function of tracer concentration. It is typically best to 
select a tracer concentration near Kd or below Kd to ensure sensitive detection of inhibitors. For example, the measured IC50 
value from a simple compound titration will approach Ki (dissociation constant of a competitive inhibitor) if [tracer] < tracer 
Kd and [kinase] << [tracer]. The majority of kinase assays validated by Invitrogen yield a robust signal with the tracer no 
more than twice the Kd value. In many cases, the Kd value can also be used to calculate Ki from a compound titration 
experiment using the Cheng-Prusoff equation (see Procedure 2), which compensates for the tracer concentration being above 
Kd. The other factor to consider when selecting a tracer concentration is the signal strength or “assay window” as it 
correlates very well with assay robustness (i.e. Z’ values). Although in many cases assay windows can exceed 10-fold, 
excellent Z’ values are typically obtained with an assay window as low as 2-fold (See Appendix A). The specific end 
application may also impact the choice of tracer concentration, based on both the assay window, Z’, and Kd value. 
 
The relatively simple method to determine tracer Kd described in Procedure 1 is supported by data from an alternative method 
to calculate Kd (in addition to Ki) as described in Appendix B. This alternative method is based on a series of inhibitor 
titrations performed at different tracer concentrations and the resulting Kd values correlate well with those derived using the 
more rapid method of Procedure 1. 
Estimation of tracer Kd and assay window 
(1.1) Reagent preparation 
1. A dilution series of tracer is prepared at 3 times the final concentration to be assayed. 
a. Dilute tracer to 3000 nM by adding 3.6 µL of 50 µM stock tracer to 56 µL of 1X Kinase Buffer A. 
b. Add 50 µL of 1X Kinase Buffer A to 6 wells in each of 2 columns of a 96-well plate. 
c. Add 50 µL of 3000 nM tracer to well A1 and mix. 
d. Remove 50 µL from well A1, transfer to well A2 and mix. 
e. Remove 50 µL from well A2, transfer to well B1 and mix. 
f. Continue process as depicted in Figure 2. 
Co
lum
n 
1
Co
lum
n 
2
A
B
C
D
E
F
Serial dilution of tracer 
in Kinase Buffer A
 
Figure 2. Serial dilution of tracer. 
 
 
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2. Prepare kinase/antibody solution at 15 nM kinase and 6 nM antibody (3X the desired final assay concentration). 
Centrifuge the antibody tube at approximately 10,000 x g for 10 minutes and aspirate desired volume from the top 
of the solution. Add the volumes of reagents calculated below to calculated volume of Kinase Buffer A. 
Calculations (for a 1000 µL solution): 
Stock kinase conc. (nM) = stock conc. (mg/mL) * 1,000,000,000 (nmol/mol) 
 kinase MW (grams/mol) 
 
Kinase volume needed (µL) = 1000 µL * 15 nM .M 
 Stock kinase conc. (nM) 
 
Antibody volume needed (µL) = 1000 µL * 6 nM . 
 Stock antibody conc. (µM) * 1000 (nmol/µmol) 
 
Kinase Buffer A needed (µL) = 1000 µL – kinase volume needed (µL) – antibody volume needed (µL) 
 
3. Prepare 30 µM staurosporine (“competitor solution”) by diluting 30 µL of 1 mM staurosporine (from a stock in 
DMSO) into 970 µL Kinase Buffer A. 
4. Prepare 3% DMSO control solution by adding 30 µL DMSO to 970 µL Kinase Buffer A. 
 
 (1.2) Experimental procedure 
1. Add 5 µL of each concentration of serially diluted tracer to six replicate assay wells in a 384-well plate (columns 
1-6) as depicted in Figure 3. 
Assay plate
(384-Well)
Assay plate
(384-Well)
Co
lum
n 
1
A
B
C
D
E
F
Co
lum
n 
2
A
B
C
D
E
F
A B
 
Figure 3. Transfer of tracer dilutions from 96-well to 384-well plate. 
 
2. Add 5 µL of competitor solution to three wells for each tracer concentration (columns 1-3). 
3. Add 5 µL of DMSO control solution to the other three wells for each tracer concentration (columns 4-6). 
4. Add 5 µL of kinase/antibody solution to all wells in columns 1-6. 
 
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5. Incubate the plate at room temperature for 60 min and read plate. 
(1.3) Data analysis 
1. Divide the acceptor/tracer emission (665 nM) by the antibody/donor emission (615 nM) to calculate the “emission 
ratio”. 
2. Plot [tracer] versus emission ratio for the competitor (staurosporine) and control (DMSO only) (Figure 4). The 
sigmoidal dose-response curve with a variable slope can be fit to the data (optional). The following equation can 
be used with GraphPadTM Prism software: 
F=50 
logEC50=logECF-(1/HillSlope)*log(F/(100-F)) 
Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) 
PIM2
0.1 1 10 100 1000
0.00
0.05
0.10
0.15
0.20
no inhibitor
10 μM staurosporine
[Kinase Tracer 236] (nM)
Em
iss
io
n 
ra
tio
 
Figure 4. Tracer titration curve. 
 
3. The assay window for any given tracer concentration can be calculated by dividing the signal in the absence of 
competitor (+ staurosporine curve) by the signal in the presence of competitor. The assay window is one of two 
criteria (the other being the tracer Kd) that are typically used to select a tracer concentration for inhibitor studies. 
Assays windows of ≥ 2 usually result in high Z’ values (see Appendix A). 
4. Subtract the competitor curve (+ staurosporine) from the control curve (DMSO only) to correct for background 
signal, which is typically due to diffusion enhanced FRET from Eu to unbound tracer. 
5. Plot the background-corrected emission ratios versus [tracer] and fit to the one site binding (hyperbola) equation to 
estimate the dissociation constant (Figure 5). In some cases data at the highest one or two tracer concentrations 
are excluded from curve fits due to relatively high “background” signal in presence of competitor. This can be 
observed as these data points deviate from the one site binding model, whereas the other points align. 
The following equation can be used with GraphPadTM Prism software: Y=Bmax*X/(Kd + X) 
 
 
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PIM2
0 50 100 150 200 250 300
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
nM320Kd =
[Kinase tracer 236] (nM)
C
or
re
ct
ed
 e
m
is
si
on
 r
at
io
 
Figure 5. Tracer Kd determination. 
 
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Procedure 2. IC50 determination 
This procedure describes how to determine inhibitor potencies by generating a 10-point IC50 curve from a 4-fold dilution 
series of test compound. The concentration of tracer used in the below protocol is based on the tracer titration from 
Procedure 1. 
 
100 nM Tracer 236 was chosen by Invitrogen for inhibitor studies. Under these conditions a high Z’ value of 0.50 was 
obtained while keeping the tracer concentration close to the Kd value (320 nM). 
 
 (2.1) Reagent preparation 
1. Prepare an intermediate dilution series of each test compound by 4X serial dilution in DMSO such that the top 
concentration is 1 mM (suggested starting point) (Figure 6, Step 1). 
a. Prepare 4 mM test compound in DMSO. 
b. Add 60 µL of DMSO to 5 wells in each of 2 columns of a 96-well plate (wells A1 to E2). 
c. Add 20 µL of 4 mM compound to well A1 and mix. 
d. Remove 20 µL from well A1, transfer to well A2 and mix. 
e. Remove 20 µL from well A2, transfer to well B1 and mix. 
f. Continue process as depicted in Step 1 of Figure 6. 
 
Co
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n 
1
Co
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n 
2
A
B
C
D
E
Co
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n 
1
Co
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n 
2
A
B
C
D
E
Co
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n 
1
A
B
C
D
E
Co
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n 
2
A
B
C
D
E
1 2 3 1 2 3
Master Dilutions
100% DMSO
4X serial dilution
100X conc.
(96-well plate)
Intermediate Dilutions
3% DMSO
3X conc.
(96-well plate)
Assay Plate
(384-well plate)
Assay Plate
(384-well plate)
STEP 1 STEP 2 STEP 3 STEP 4
 
Figure 6. Compound serial dilution. 
 
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2. Dilute the “Master Dilution” series 33.3-fold into Kinase Buffer A. Remove 5 µL of each concentration of diluted 
compound, transfer to another 96-well plate, add 162 µL of Kinase Buffer A and mix (Figure 6, Step 2). 
3. Prepare tracer solution in Kinase Buffer A at 300 nM tracer (3X the desired final assay concentration). Add the 
volumes of reagents calculated below to calculated volume of Kinase Buffer A. 
Calculations (for a 1000 µL solution): 
 
Tracer volume needed (µL) = 1000 µL1 * 300 nM2 .M 
 1000 nM/µM * 50 µM3 
 1final volume of solution 
 2desired 3X tracer concentration 
 3stock tracer concentration 
 
Kinase Buffer A needed (µL) = 1000 µL – tracer volume needed (µL) 
 
4. Prepare kinase/antibody solution at 15 nM kinase and 6 nM antibody (3X the desired final assay concentration). 
Centrifuge the antibody tube at approximately 10,000 x g for 10 minutes and aspirate desired volume from the top 
of the solution. Add volumes of reagents calculated below to calculated volume of Kinase Buffer A. 
Calculations (for a 1000 µL solution): 
Stock kinase conc. (nM) = stock conc (mg/mL) * 1,000,000,000 (nmol/mol) 
 kinase MW (grams/mol) 
 
Kinase volume needed (µL) = 1000 µL * 15 nM . 
 Stock kinase conc. (nM) 
 
Antibody volume needed (µL) = 1000 µL * 6 nM . 
 Stock antibody conc (µM) * 1000 (nmol/µmol) 
 
Kinase Buffer A needed (µL) = 1000 µL – kinase volume needed (µL) – antibody volume needed (µL) 
 
 (2.2) Experimental procedure 
1. Add 5 µL of each concentration of serially diluted compound to triplicate assay wells in a 384-well plate (columns 
1-3) as depicted in Steps 2 and 3 of Figure 6. 
2. Add 5 µL of kinase/antibody solution to all wells. 
3. Add 5 µL of tracer solution to all wells. 
4. Incubate the plate at room temperature for 60 min and read. 
Note: 60 minutes is a general guideline for incubation. However, in some cases multiple read times or continuous 
measurements may be used to examine the kinetics of binding reactions as might be of interest for studies on slow-
binding compounds. 
 
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(2.3) Data analysis 
1. Divide the acceptor/tracer emission (665 nM) by the antibody/donor emission (615 nM) to calculate the “emission 
ratio”. 
2. Plot [test compound] versus emission ratio. The sigmoidal dose-response curve with a variable slope can be fit to 
the data (see section 1.3, step 2 for equation). Representative date generated by Invitrogen for a set of well-
characterized kinase inhibitors is presented in Figure 7. A comparison of IC50 values to literature and in some 
cases internally generated data from activity-based assays (SelectScreenTM profiling service) are provided as a 
reference in Table 1. 
3. In some cases, the Cheng-Prusoff equation1 (equation 1) can be used to convert IC50 to Ki based on the 
concentration of Tracer and an accurate Tracer Kd (determined by following Procedure 1 or Appendix B). 
(1) 
( )
[ ]
⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛
⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
+
=
d
i
K
Tracer
ICK
1
50 
 
This relationship holds true when the following criteria are met: 
1. [kinase] << [tracer] and [kinase] << IC50 
2. [kinase] < Tracer Kd 
3. There is a single class of binding sites 
In order to determine Ki values for very tight-binding compounds, it may be necessary to perform assays at lower 
kinase concentrations. 
 
1Cheng, Y.C., Prusoff, W.H. Biochem Pharmacol. (22) 3099-3108 (1973). 
 
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PIM2
0.1 1 10 100 1000 10000
0.25
0.26
0.27
0.28
0.29
0.30
0.31
0.32
0.33
0.34
0.35
0.36
0.37
0.38
0.39
0.40
0.41
0.42
0.43
0.44
0.45
0.46
Staurosporine
Dasatinib
PP2
Imatinib
VX680
Sunitinib
Gefitinib
Sorafenib
[compound] (nM)
Em
is
si
on
 ra
tio
 
Figure 7. Representative data generated at Invitrogen. 
 
 
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Table 1. IC50 values (nM) and comparison to literature and activity-based data. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Karaman, M.Z., et al. Nat. Biotechnol. 26(1) 127-132 (2008) 
 
 
Note: These data are provided for references purposes. It is important to consider that the source of enzymes 
and method of detection (activity assay vs. binding assay) will affect whether measurements of IC50 values are 
due to active kinase, non-activated kinase, or a combination of both. 
Compound 
LanthaScreenTM Eu 
Kinase Binding Assay 
SelectScreenTM 
Kinase Profiling Service 
 
Literature Kd values 
(Karaman et al.) 
Staurosporine 6.9 41 1.9 
Dasatinib >10000 >5000 >10000 
PP2 >10000 >5000 n.d. 
Imatinib >10000 >5000 >10000 
VX680 >10000 >5000 >10000 
Sunitinib 4800 >5000 >10000 
Gefitinib >10000 n.d. >10000 
Sorafenib >10000 >5000 >10000 
 
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Appendix A. Assay robustness as a function of assay window 
 
The Z’-factors for 215 kinase:tracer pairs were determined and plotted as a function of assay window (Figure A1). The data 
demonstrate excellent Z’ values are typically obtained with an assay window of ≥ 2. Whereas, assay windows in the 1.5 to 2-
fold range yield Z’ values between 0.4 and 0.7, which may be suitable for some applications. 
0 5 10 15 20 25 30
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Assay Window
Z'
-f
ac
to
r
 
Figure A1. Z’-factor as a function of assay window for 215 kinases. 
 
 
 
 
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Appendix B. Alternate method to determine tracer Kd and Ki values 
 
A simple method to determine tracer Kd values from a tracer titration is described in Procedure 1, whereas an alternate 
method is described here based on IC50 curves performed at various tracer concentrations followed by analysis with the 
Cheng-Prusoff equation. In addition to determination of tracer Kd values, this method also enables calculation of Ki values 
(dissociation constant for the inhibitor). Rearrangement of the Cheng-Prusoff equation results in a linear relationship (i.e. the 
form y = mx + b) useful for analysis of binding data from homogenous assays (equation 2)2. When plotted with the IC50 
value on the y-axis and the tracer concentration of the x-axis, the Ki is equal to the y-intercept and the slope equals [Ki]/[Kd]. 
Thus, the y-intercept divided by the slope equals the tracer Kd. This method enables calculation of the tracer Kd from IC50 
curves performed at various concentrations of tracer. 
 (2) [ ] i
d
i KTracer
K
KIC +⎟⎟
⎠
⎞
⎜
⎜
⎝
⎛
×⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
=50 
 
Application of the Cheng-Prusoff equation is valid if the following criteria are met: 
1. There is a single class of ligand binding site 
2. The is no ligand depletion (i.e. [tracer] >> [kinase]) 
3. The receptor concentration < Kd 
 
This method was applied to calculated the tracer Kd for representative kinase:tracer interactions with a range affinities and 
compared to the tracer titration method (Procedure 1). Example data are presented for the kinase TEK. The Kd value 
calculated from a tracer titration is 29 nM whereas that calculated using the linearized Cheng-Prusoff equation using 
staurosporine as the inhibitor is 31 nM and using VX680 is 30 nM, in close agreement (Figure B1). Data for all kinases 
compared with both methods is in Table B1, showing close agreement between both methods and supporting use of the more 
simple method based on a single tracer titration. 
 
2Newton, P., et al. J Biomol Screen. 13(7) 674-682 (2008). 
 
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A 
0 25 50 75 100 125 150
0.0
0.1
0.2
0.3
0.4
TEK/PV3628
Kd = 29 nM
[Tracer 236] nM
C
or
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ct
ed
 e
m
is
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tio
 
 
B C 
 
 
D E 
 
Figure B1. Determination of tracer Kd values by linearized Cheng-Prusoff equation and tracer titration method. The 
tracer Kd value was determined by the tracer titration method essentially as described in Procedure 1 with Kinase Tracer 236 
and Eu-anti-GST antibody (A). IC50 curves were determined for TEK for the inhibitors staurosporine (B) and VX680 (C) 
essentially as described in Procedure 2 with various concentrations of Kinase Tracer 236. IC50 values were then plotted 
against the tracer concentration and the Kd values for the Tracer and Kinase and the Ki values for the inhibitor and the Kinase 
were determined from the slope and y-intercept. 
TEK Staurosporine IC50 plot
0 50 100 150 200 250 300
0
50
100
150
200
250
300
Kd for Tracer 236 = 31 nM
Ki for Staurosporine = 28 nM
[Kinase Tracer 236] nM
IC
50
 v
al
ue
TEK VX680 IC50 plot
0 50 100 150 200 250 300
0
100
200
300
400
500
600
700
800
900
1000
1100
Kd for Tracer 236 = 32 nM
Ki for VX680 = 120 nM
[Kinase Tracer 236] nM
IC
50
 v
al
ue
TEK/PV3628
Varying Tracer 236 concentrations
Staurosporine titration
0.1 1 10 100 1000 10000 100000
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
250 nM
125 nM
63 nM
32 nM
16 nM
8 nM
4 nM
2 nM
1 nM
0.5 nM
[Staurosporine] nM
Em
is
si
on
 r
at
io
[tracer]
TEK/PV3628
Varying Tracer 236concentrations
VX680 titration
0.1 1 10 100 1000 10000
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
250 nM
125 nM
63 nM
32 nM
16 nM
8 nM
4 nM
2 nM
1 nM
0.5 nM
[VX680] nM
Em
is
si
on
 r
at
io
[tracer]
 
LanthaScreen™ Eu Kinase Binding 
Assay Validation Packet 
Version No.: 
1  Page 17 of 17 
Optimization of a LanthaScreen™ Eu Kinase Binding Assay for PIM2 
 
 
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Table B1. Comparison of Kd determination by linearized Cheng-Prusoff equation and tracer titration method. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Tracer Kd values (nM) 
Kinase Tracer titration Linearized Cheng-Prusoff
with Staurosporine 
Linearized Cheng-Prusoff 
with VX-680 
TEK 29 31 30 
TAOK2 60 82 71 
ITK 46 45 n.d. 
MAP3K3 184 230 n.d. 
MYLK2 237 299 n.d.